Chemiluminescence is the generation of light as a product of certain chemical reactions, typically conducted at ordinary or room temperature. These reactions typically produce a reaction intermediate in an electronically excited or active state; the radioactive decay of the excited state to the ground state is the source of visible light, or luminescence in the form of visible light, ultraviolet, or infrared radiation. Most importantly, the formation of electronically excited reaction products can be detected by measuring the luminescence intensity, even when the radiation is minimal or negligible.
Most chemiluminescent reactions can be classified into (1) peroxide decomposition, such as bioluminescence or peroxyoxalate chemiluminescence; (2) singlet oxygen chemiluminescence; and (3) ion radical or electron transfer chemiluminescence, such as electrochemiluminescence. In principle, one molecule of a chemiluminescent reactant can react to produce one electronically excited molecule, which in turn can emit one photon of light. Light yields can therefore be defined in the same terms as chemical product yields, i.e., in units of einsteins of light emitted per mole of chemiluminescent reactant.
Electrogenerated chemiluminescence, alternatively referred to as electrochemiluminescence (ECL), is an electrochemical means for converting electrical energy into light. With ECL processes, controlling the electrode potential is an extremely powerful control mechanism for modulating the rate of light generating reactions, a characteristic that provides certain advantages over chemiluminescence or bioluminescence methods. For example, ECL has been used with great success to image the heterogeneity electron-transfer rates at electrode surfaces with submicrometer spatial resolution.
ECL systems and devices typically use solutions of luminescent molecules, i.e. molecules which upon electrical excitation are capable of emitting ECL, such as luminol, ruthenium tris (2,2'-bipyridine) dichloride hexahydrate Ru(bpy).sub.3 Cl.sub.2 .cndot.6H.sub.2 O!, or Ru(bpy).sub.3.sup.2+. ECL processes have been demonstrated for many different molecules by several different mechanisms. For example, in one of the many electrochemiluminescence reactions, alternating current may be used to excite hydrocarbon radicals. The reaction intermediates, anions and cations, are continuously formed and destroyed, and in doing so, release radiant energy. It is believed that the energy released as a product of an electron transfer process produces the light. Since the lifetimes of excited luminol and Ru(bpy).sub.3.sup.2+ are on the microsecond time scale, the excited molecules cannot diffuse more than a few hundred nanometers before emitting their photons.
ECL has been developed as a highly sensitive process in which reactive species are generated from stable precursors (i.e., the ECL-active label) at the surface of an electrode. This technology has many distinct advantages over other detection systems: no radioisotopes are used; detection limits for the label are extremely low (200 fmol/L); the dynamic range for label quantification extends over six orders of magnitude; the labels are extremely stable compared with those of most other chemiluminescent systems; the labels, small molecules (.about.1000 Da), can be used to label haptens or large molecules, and multiple labels can be coupled to proteins or oligonucleotides without affecting immunoreactivity, solubility, or ability to hybridize; because the luminescence is initiated electrochemically, selectivity of bound and unbound fractions can be based on the ability of labeled species to access the electrode surface, so that both separation and nonseparation assays can be set up; and measurement is simple and rapid, requiring only a few seconds.
A typical ECL cell includes a first electrode and a second electrode, a first compound capable of generating ECL, a second compound capable of reacting with the first compound to generate ECL, and a source of supply of an electrical potential between the electrodes. In conventional ECL devices, the luminophores are homogeneously distributed in solutions, a fluidic pathway, or polymer films, or the active constituents (i.e., the luminescent molecules) may be confined to the surface of the electrodes as immobilized layers.
As an electrical potential is supplied to the first electrode, the first compound is electrolyzed to produce a species, a reaction intermediate, that reacts with a second compound (dissolved in a solution) to generate ECL. The ECL emission from the luminophore can be easily detected and is very intense with some electrode materials. The intensity of the luminescence increases with the applied voltage until the reactant species at the surface of the electrode is depleted, resulting in decreased intensity. The intensity of the observed luminescence is great enough that it can easily be measured with conventional transducers, such as a photomultiplier tube (PMT), operating either in photon-counting or current modes.
ECL may be used in a wide variety of detection protocols. For example, immunoassays have been readily demonstrated and developed into a wide range of different formats. Nonseparation competitive assays of haptens can be formatted by using a labeled hapten that competes for antibody with the analyte. Competitive assays for either haptens or large molecules can also be formatted as solid-phase assays. Microparticles coated with antigen compete with the analyte for labeled antibody. This assay format can be used for either large analytes or haptens. Solid-phase sandwich immunoassays can also be formatted by using two antibodies specific for different epitopes of the analyte. These solid-phase assays can be formatted as either separation or nonseparation assays. DNA probe assays can also be performed with use of ECL to detect hybridization of labeled probes to nucleic acid sequences. All such measurements typically require the use of a luminometer, if the sample is in a liquid, or an imaging device, if the light is emitted from a two-dimensional sample, e.g. a membrane used in blotting.
The luminometer is designed to measure small amounts of light, usually in the visible spectrum. The sample is presented in a cuvette, the volume of sample being usually between 100 and 500 .mu.l and the cuvette (usually 10-12 mm diameter and about 40-75 mm long and disposable, being made from polycarbonate or similar transparent plastic) capable of holding up to 3 ml. Light is detected by a photomultiplier or by a solid state photodiode. Signals from the detectors may be either current or photon-counts. The instrument may have the capability to handle automatically many samples in a programmed or programmable fashion. Instruments may be fitted with automatic injectors so that several reagents may be added to the sample during the course of processing. A few instruments have temperature control for the sample and some incorporates a temperature compensation factor to correct the result. Most units are fitted with a RS232 port so that the luminometer may be connected to, and in some instances controlled by, an external computer.
ECL processes exhibit an extremely wide response in intensity to luminophore concentration ranges. The typical range is about 10.sup.6 to about 10.sup.7 counts. To provide proper results, the luminometer detection range preferably equals or exceeds the range for the ECL process (and assay range) . Commercially available luminometers, typically provided with one gain setting, are not always capable of meeting the necessary range. If the sample intensity is over-range (greater than the range for the luminometer, e.g., greater than 10.sup.7 counts) or under-range (to little), the reading is lost and must be re-run, costing time and reagent expense. Since one reading per sample is generated, if a reading goes off-scale it cannot be determined unless the run is repeated successfully at a lower luminometer gain or sample dilution. Furthermore, conventional ECL protocols using conventional equipment are typically a compromise between the low end amount of luminescence and the high end, since the luminometer has a fixed range at only one gain setting.
Thus, there is a need to improve the dynamic range for ECL detection and to eliminate the need for repeating the process.